Dissecting molecular evolution of class 1 integron gene cassettes and identifying their bacterial hosts in suburban creeks via epicPCR

Abstract

Abstract Objectives Our study aimed to sequence class 1 integrons in uncultured environmental bacterial cells in freshwater from suburban creeks and uncover the taxonomy of their bacterial hosts. We also aimed to characterize integron gene cassettes with altered DNA sequences relative to those from databases or literature and identify key signatures of their molecular evolution. Methods We applied a single-cell fusion PCR-based technique—emulsion, paired isolation and concatenation PCR (epicPCR)—to link class 1 integron gene cassette arrays to the phylogenetic markers of their bacterial hosts. The levels of streptomycin resistance conferred by the WT and altered aadA5 and aadA11 gene cassettes that encode aminoglycoside (3″) adenylyltransferases were experimentally quantified in an Escherichia coli host. Results Class 1 integron gene cassette arrays were detected in Alphaproteobacteria and Gammaproteobacteria hosts. A subset of three gene cassettes displayed signatures of molecular evolution, namely the gain of a regulatory 5′-untranslated region (5′-UTR), the loss of attC recombination sites between adjacent gene cassettes, and the invasion of a 5′-UTR by an IS element. Notably, our experimental testing of a novel variant of the aadA11 gene cassette demonstrated that gaining the observed 5′-UTR contributed to a 3-fold increase in the MIC of streptomycin relative to the ancestral reference gene cassette in E. coli. Conclusions Dissecting the observed signatures of molecular evolution of class 1 integrons allowed us to explain their effects on antibiotic resistance phenotypes, while identifying their bacterial hosts enabled us to make better inferences on the likely origins of novel gene cassettes and IS that invade known gene cassettes.